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Biochemistry 2005, 44, 12214-12228
PsbU Provides a Stable Architecture for the Oxygen-Evolving System in Cyanobacterial Photosystem II† Natsuko Inoue-Kashino,‡,§,| Yasuhiro Kashino,*,‡,| Kazuhiko Satoh,| Ichiro Terashima,§ and Himadri B. Pakrasi‡ Department of Biology, Washington UniVersity, St. Louis, Missouri 63130, Department of Biology, Graduate School of Science, Osaka UniVersity, Toyonaka, Osaka 560-0043, Japan, and Department of Life Science, Graduate School of Life Science, UniVersity of Hyogo, Ako-gun, Hyogo 678-1297, Japan ReceiVed NoVember 22, 2004; ReVised Manuscript ReceiVed July 14, 2005
ABSTRACT: PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], we investigated the function of PsbU using a psbU deletion mutant (∆PsbU) of Synechocystis 6803. In contrast to the wild type, ∆PsbU did not grow when both Ca2+ and Cl- were eliminated from the growth medium. When only Ca2+ was eliminated, ∆PsbU grew well, whereas when Cl- was eliminated, the growth rate was highly suppressed. Although ∆PsbU grew normally in the presence of both ions under moderate light, PS II-related disorders were observed as follows. (1) The mutant cells were highly susceptible to photoinhibition. (2) Both the efficiency of light utilization under low irradiance and the chlorophyll-specific maximum rate of oxygen evolution in ∆PsbU cells were 60% lower than those of the wild type. (3) The decay of the S2 state in ∆PsbU cells was decelerated. (4) In isolated PS II complexes from ∆PsbU cells, the amounts of the other three lumenal extrinsic proteins and the electron donation rate were drastically decreased, indicating that the water oxidation system became significantly labile without PsbU. Furthermore, oxygen-evolving activity in ∆PsbU thylakoid membranes was highly suppressed in the absence of Cl-, and 60% of the activity was restored by NO3- but not by SO42-, indicating that PsbU had functions other than stabilizing Cl-. On the basis of these results, we conclude that PsbU is crucial for the stable architecture of the water-splitting system to optimize the efficiency of the oxygen evolution process.
The photosynthetic electron transport system in oxygenic photosynthesis converts light energy to biochemical energy and consists of three major large membrane protein complexes, namely, photosystem I (PS I),1 cytochrome (cyt) b6f, and photosystem II (PS II) complexes (1). Recently, all of these complexes were crystallized, and the structural models were presented at high resolution [2.5 Å in cyanobacterial PS I (2), 4.4 Å in plant PS I (3), 3.0 and 3.1 Å in cyt b6f (4, 5), and 3.2-3.8 Å in PS II (6-9)]. Among these complexes, PS II is the most integrated membrane protein complex, † This work was supported by funding from the NSF (MCB0215359, H.B.P.) and grants from the Hyogo Prefecture (Y.K.) and the 21st Century Center of Excellence Program (COE) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (I.T. and Y.K.). * Address correspondence to this author at the University of Hyogo. Phone/Fax: +81 791 58 0185. E-mail:
[email protected]. ‡ Washington University. § Osaka University. | University of Hyogo. 1 Abbreviations; R, initial slope in light curve; Chl, chlorophyll; CP47 and CP43, peripheral Chl binding proteins of 47 and 43 kDa in PS II; cyt, cytochrome; DCBQ, dichloro-p-benzoquinone; DCMU, 3-(3,4dichlorophenyl)-1,1-dimethylurea; F0, Fm, and Fv, initial, maximal, and variable fluorescence yield; Ik, intensity at the onset of light saturation; Kmr, Gmr, and Emr, kanamycin-, gentamycin-, and erythromycinresistance cartridges, respectively; PS I and PS II, photosystem I and II; MES, 2-(N-morpholino)ethanesulfonic acid monohydrate; Pmax, maximum photosynthetic rate.
which consists of over 20 distinctive membrane proteins, three lumenal extrinsic proteins, and several cofactors (6, 8, 10, 11). On the basis of this structure, PS II extracts electrons from water molecules and exhausts molecular oxygen. Although both cyanobacterial and plant PS II complexes contain three lumenal extrinsic proteins that play important roles in PS II-mediated water oxidation, only one of them (PsbO) is shared in both complexes; PsbO, PsbU, and PsbV in cyanobacterial PS II and PsbO, PsbP, and PsbQ in plant PS II (10). Until now, no similarity was found in the amino acid sequences and structures between the residual two proteins (PsbU and PsbV vs PsbP and PsbQ) (6, 11-13). PsbU was first found as a 9 kDa lumenal extrinsic protein in a highly active PS II complex isolated from Phormidium laminosum, which assisted oxygen evolution with PsbO manganese-stabilizing protein (14, 15). Another peripheral protein, cytochrome c-550, was found in Anacystis nidulans and Microcystis aeruginosa (16, 17) and was attributed later as an important lumenal extrinsic protein of cyanobacterial PS II (PsbV) (18, 19). Both of the corresponding genes (psbU and psbV) were then found in all cyanobacterial genomes analyzed except for two strains of Prochlorophytes, Prochlorococcus marinus MED4 and P. marinus SS120 (20). Genetic and biochemical investigations on these two proteins have been performed in the past decade. Shen et al. genetically deleted psbU and/or psbV genes from the
10.1021/bi047539k CCC: $30.25 © 2005 American Chemical Society Published on Web 08/19/2005
PsbU Is Necessary for Stable Oxidizing Side of PS II cyanobacterium Synechocystis sp. PCC 6803 and investigated the effects of these deletions (21, 22). Without PsbV, the cells did not grow autotrophically when Ca2+ or Cl- was eliminated from the growth media. Furthermore, the psbV deletion mutant (∆PsbV) showed unusual S-state transitions and exhibited an unstable oxygen-evolving activity (22). Although Shen et al. also detected modification of the S2 state in the psbU deletion mutant (∆PsbU) by examining thermoluminescence, they did not observe a remarkable impairment in the growth rates under any growth conditions tested (normal BG11, BG11-Ca2+, BG11-Cl-) (21, 22). On the basis of these results, they concluded that PsbV was important for the stability and function of the manganese cluster in cyanobacterial PS II, while they only suggested that PsbU would play roles in maintaining the normal S-state transitions and in the maximum affinity of PS II for Ca2+ and Cl- ions (21, 22). Similar to the extrinsic proteins in higher plant PS II (PsbO, PsbP, and PsbQ), PsbU and PsbV can also be removed from isolated PS II by high concentrations of salts (11, 18, 19). Using isolated cyanobacterial and red algal PS II complexes, Shen and his group demonstrated that, without PsbU or PsbV, PS II complexes require high concentrations of Ca2+ and Cl- for the high oxygen-evolving activity (19, 23). PsbV can be reconstituted alone to the salt-washed PS II complexes, whereas the assembly of PsbV and PsbO to the salt-washed PS II is a prerequisite for the effective reconstitution of PsbU (19). With regard to the binding characteristics, PsbU and PsbV correspond to PsbQ and PsbP in higher plant PS II (24). On the basis of these biochemical analyses, it is generally accepted that the entities and functions of PsbU and PsbV proteins were replaced with PsbQ and PsbP proteins, respectively, during the evolutional process (18, 19, 25, 26). Recently, we have found two proteins in the cyanobacterial PS II complexes from Synechocystis 6803 which have homology to plant PsbP and PsbQ (sequence identities are 27.3% and 32.9% for PsbP and PsbQ between Synechocystis 6803 and Arabidopsis, respectively) (11, 27) although these proteins are not found in the reported three-dimensional PS II structure from thermophilic cyanobacteria (6-9). Genome information also supports the presence of PsbP and PsbQ homologues in several cyanobacteria (20, 27). In addition, Summerfield et al. (28) demonstrated the association of the PsbQ homologue to the PS II complex and the importance of this protein for PS II stability in Synechocystis 6803 (28), and Ohta et al. reported the presence of a protein homologous to plant PsbQ in a primitive red alga, Cyanidium caldarium (29). These findings of PsbP and PsbQ in cyanobacteria and PsbQ in red algae throw doubt on the above-mentioned hypothesis that PsbU and PsbV were functionally and physically replaced by PsbP and PsbQ during the evolutional process to plant chloroplasts (25). Therefore, the functions of PsbU and PsbV in relation to those of PsbQ and PsbP should be reexamined. In this study, we conducted detailed analyses to clarify the function of PsbU using the deletion mutant of the psbU gene (∆PsbU). Since Ca2+ and Cl- are well-known to be essential for oxygen evolution, we, first, examined the growth of cells under the Ca2+- and/or Cl-depleted conditions. The Cl- depletion resulted in a severer suppression of growth rate in ∆PsbU cells than the absence of Ca2+. We also found that ∆PsbU cells were more
Biochemistry, Vol. 44, No. 36, 2005 12215 susceptible to photoinhibition. The fluorescence rise kinetics showed that water oxidation in PS II was slowed in ∆PsbU mutant cells grown in normal BG11 and further if they were grown without Cl-. Light curves exhibited that the efficiency of oxygen evolution in ∆PsbU cells was remarkably lower than that of the wild type. The lifetime of the S2 state in ∆PsbU cells grown in normal BG11 was prolonged. The absence of PsbU did not affect the stoichiometry of PS II and PS I. In isolated thylakoids, oxygen-evolving activity was highly suppressed, and part of the activity was restored by the addition of monovalent anion, NO3-. In the PS II complexes isolated from ∆PsbU cells by highly mild conditions, the amounts of the PsbO, PsbQ, and PsbV proteins and the electron donation rate were drastically decreased. Unlike the original proposal for PsbU function (10) as affinity sites for Ca2+ and/or Cl-, the present results clearly indicate that PsbU is important in construction of a stable architecture of the oxygen-evolving system in PS II. MATERIALS AND METHODS Cyanobacterial Strains and Growth Conditions. Synechocystis 6803 cells (the wild type and the mutants which lacked PsbU or PsbO) were grown on a rotary shaker at 30 °C under 50 µmol of photons‚m-2‚s-1 of white light in BG11 medium (30) using plastic tissue culture plates with 12 wells (CELLSTAR; Greiner Bio-One, Frickenhausen, Germany) with 3 mL triplicates for each medium condition to measure the growth rates. The cells were grown with bubbling air in plastic tissue culture flasks [250 mL (75 cm2); Corning, Corning, NY] for the measurement of oxygen evolution. Prior to growth measurements, cells that were cultivated in normal BG11 for 3 days were washed three times with CaCl2depleted BG11 medium (BG11-CaCl2) and resuspended in the same medium. They were then diluted into the appropriate media as described below to OD730nm ) 0.05-0.06. Ca2+- and/or Cl--depleted BG11 medium was prepared in plastic ware to exclude contamination of ions. Media were prepared with either Ca2+ [240 µM Ca(NO3)2] or Cl- (480 µM NaCl). Growth was monitored by measuring OD730nm on a MQX200 µQuant Universal microplate spectrophotometer with operating software KC4 (Bio-Tek Instruments, Winooski, VT) and 96-well tissue culture plates (150 µL from each sample). The accuracy of this monitoring system was checked in advance by a DW2000 spectrophotometer (SLM-Aminco, Urbana, IL). Gene Cloning and Isolation of Mutants. The upstream and downstream regions [298488-297889 and 297469-296961 (Cyanobase, http://www.kazusa.or.jp/cyanobase/)] of the sll1194 (psbU) gene of Synechocystis 6803 were amplified by PCR using the following oligonucleotide primers (Figure 1A): 5′-CGAAGCTTTGGAAAATAATG-3′ (primer P1) and 5′-GAATTGTCTCCTATAACCAA-3′ (primer P2) for 298488-297889 and 5′-GCGCGGAGCTCCATTATTCATTGTCTAGATA-3′ (primer P3) and 5′-GCGCGGAGCTCTTTTGATCAGTCATTTGGAA-3′ (primer P4) with SacI restriction sites (underlined sequences in the primers) for 297469-296961. The PCR product from the upstream region (#1 in Figure 1) was cloned into TA cloning vector pGEM-T (Promega, Madison, WI) (Figure 1B). Then, the PCR product from the downstream region (#2 in Figure 1), which had been processed by SacI, was inserted into the SacI site of
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FIGURE 1: Scheme for making constructs to delete and complement the psbU gene (A-E). The solid bars which are expressed by #1, #2, and #5 are the PCR products from genome DNA, and solid bars #3, #4, and #6 are the antibiotic-resistance cartridges. Kmr, Gmr, and Emr are the kanamycin-, gentamycin-, and erythromycin-resistance cartridges, respectively. Segregation of the deletion and complementation mutations of the psbU gene was examined by PCR (F). The PCR products obtained with the P1 and P4 primers using genomic DNA from Synechocystis 6803 cells were subjected to electrophoresis. Lanes: 1, 1 kb DNA ladder (Promega, Madison, WI); 2, wild type (1.53 kb); 3, ∆PsbU (2.43 kb); 4, ∆PsbU complement (2.97 kb); 5, ∆PsbU/HT3 (2.04 kb); 6, ∆PsbU/HT3 complement (2.97 kb). Numbers in parentheses are the expected lengths of PCR products. For details, see Materials and Methods.
the resulting plasmid (Figure 1B). The orientation of these two inserts was checked by PCR using the above primers
and cleavage by several appropriate restriction enzymes (data not shown). A kanamycin-resistance cartridge (31) (Kmr, #3
PsbU Is Necessary for Stable Oxidizing Side of PS II in Figure 1) or a gentamycin-resistance cartridge (32) (Gmr, #4 in Figure 1) was introduced into the PstI site or SalI site of the resulting plasmid, respectively, which was located between the two inserts (Figure 1B). These two constructs (Figure 1C) were used to delete the psbU gene from the wildtype and HT3 (Kmr) strains of Synechocystis 6803, respectively. The HT3 strain, which has a hexahistidine tag on the carboxyl terminus of CP47 (33), was a generous gift from Prof. Terry M. Bricker at Louisiana State University. The Kmr cartridge in the HT3 strain had been inserted into the genomic DNA to select the intended mutation to generate the histidine tag (33). The ∆PsbU/wild-type strain which carries the Kmr cartridge was used from Figure 2 through Figure 6 and all figures in Supporting Information, and the ∆PsbU/HT3 strain which carries both the Kmr and Gmr cartridges was used in Figures 7 and 8. To complement the deletion of the psbU gene, the region including the psbU gene was amplified by PCR using primers P1 and P4 (Figure 1D). This region (#5 in Figure 1D) was cloned into the TA cloning vector pGEM-T (Figure 1E). The erythromycinresistance cartridge (34) (Emr, #6 in Figure 1) was then inserted into the XbaI site at 38 bp downstream of sll1194 in the resulting plasmid (Figure 1E). Accordingly, the complemented strain from ∆PsbU/wild-type carries the Emr cartridge. This complemented strain was used in Figure 5. The ∆PsbO mutant was engineered by inserting a spectinomycin-resistance cassette in a BamHI site of the psbO (sll0427) gene of Synechocystis 6803. The accuracy of all PCR products used in Figure 1 was checked by sequencing. The complete segregation of the mutation was demonstrated by PCR (Figure 1F). Isolation of Thylakoid Membranes and PS II Complexes. Thylakoid membranes from wild-type and ∆PsbU strains and the PS II complexes from the HT3 and ∆PsbU/HT3 strains were isolated as previously described (11). Thylakoid membranes were also prepared with or without Cl- ions from wild-type and ∆PsbU stains. The cells were broken by glass beads as was described in ref 11, and then the membranes were washed twice by a solution containing 50 mM 2-(Nmorpholino)ethanesulfonic acid monohydrate (MES)-NaOH (pH 6.5), 10 mM MgCl2, 5 mM CaCl2, and 1 M betaine or a solution containing 50 mM MES-NaOH (pH 6.5), 10 mM MgSO4, 5 mM CaSO4, and 1 M betaine. The resulting thylakoid membranes were resuspended in the respective solutions. The measured Cl- concentration was 36.4 mM for the former solution and less than the detectable limit (
